In 1975, a leak in the jet fuel tank of a U.S. Air Force airplane contaminated the soil in Charleston, North Carolina. More than 300,000 liters of the chemical toluene leaked into the soil, reaching the underground water supply. By 1985, the contamination had spread to many residential areas. The removal of the polluted soil was technically impossible, and the removal of just the contaminated water would not solve the problem; it was impossible to eliminate the source of pollution, the soil. One possible solution was to use naturally occurring microorganisms in the soil that have the capability to digest toluene, transforming it into carbonic gas, water, and energy (Figure 11-1).

Figure 11-1. Different bacteria have the capability to transform different substances into energy for their growth.

In 1992, bioremediation was used in Charleston, North Carolina, to clean the environment. Scientists added nutrients to stimulate growth and activity of specific microorganisms that would degrade the fuel. The nutrients were applied in the contaminated soil through infiltration tubes, and the polluted water was removed from several artesian wells. About a year later, the level of contamination was reduced by 75 percent. Close to the infiltration tubes, where the population of the bacteria had higher growth rates, the previously high levels of toluene had been reduced to undetectable levels.

Bacteria were also used for cleaning the Alaskan coast in 1989, after the infamous Exxon Valdez oil tanker ran aground, spilling tons of crude oil (Figure 11-2). Most of the viscous oil was removed from the sea by suction and filtration of the superficial layer of oil and water. However, the oil that penetrated rocks and gravel along the beaches was cleaned by bacteria that had the ability to decompose crude oil.

Source: Tanker photo courtesy of Damage Assessment and Restoration Program, National Oceanic and Atmospheric Administration.

Figure 11-2. Oil pollution has an enormous impact on the environment.

The bacteria used for cleaning the Exxon Valdez spill used oil, a hydrocarbon, as source of energy for their growth, decomposing it into smaller nontoxic compounds. Various bacteria possess different abilities to decompose residues that are toxic to man. The diversity of microbes is enormous. Whereas some microorganisms survive by feeding off of other living cells, many others use decomposing organic matter for their survival. There are also many that are able to use toxic substances as a source of energy. Some microorganisms have very eclectic metabolic pathways that allow them to use solvents such as chlorine, a typical pollutant in highly industrialized areas, as a source of energy. Those microorganisms can use chlorine as an oxidant when oxygen is not available.

Several research groups have identified bacteria that can degrade agro-chemicals and chemical fertilizers. This is additional evidence of the importance of the preservation of biodiversity. A seemingly useless microorganism in an environment might eventually be used for cleaning that environment. With the development of biotechnology, microorganisms are drawing attention from scientists and biotechnology companies. The invisible army of microorganisms that continually promotes recycling in the Earth is acquiring a new prestige.

Experience with bacteria seems to indicate that the microorganisms can use practically any substance as “food” or a source of energy. Although some materials are highly toxic for a certain bacterium, they can be a substrate for another. Bacteria have been isolated that are able to feed on detergents, sulfur, methane, chlorine, carbon tetrachloride, toluene, and other substances. The great biodiversity of microorganisms on Earth is a largely untapped reservoir of species with unexpected abilities.

Methylene chloride is considered one of the most serious pollutants because of its carcinogenic properties. This substance is produced in large amounts in certain industrial processes. It is, however, decomposed into water, carbonic gas, and salt when treated in bioreactors with a species of bacteria that decomposes the methylene chloride using enzymes to convert the chemical into energy and other nonpollutant substances. The discovery of microorganisms for cleaning the environment, in general, occurs in places where there is pollution. Microorganisms found growing in those places are at the very least resistant to pollutants occurring there. Bioremediation typically consists of harvesting many microorganisms from polluted sites to select those with the most efficient degradation abilities. They are then isolated, multiplied, and reintroduced into the areas to be cleaned. In addition to the microorganisms, some nutrients, such as nitrates and phosphates, are added to the inoculum to promote fast growth and enhanced cleaning. Depending on the nature and extension of the pollution, the bioremediation can take from a few months to many years to work. When the toxic substance is eliminated, the population of the microorganism is also reduced. Eventually, other populations of bacteria will find favorable conditions of substrate, temperature, and humidity, and will be able to grow.

One of the largest hurdles with bioremediation is the difficulty in controlling the factors for the microorganism growth. The development of a microorganism is affected by temperature, pH, humidity, and availability of an energy source. It is relatively simple to culture bacteria in a laboratory under controlled conditions, but outside in changing environmental conditions, the process is more difficult. To assist in this effort, biotechnology is establishing a constructive partnership with bioremediation to engineer more efficient microorganisms that are less dependent on environmental conditions. For instance, transferring genes from thermophilic (heat-loving) bacteria to one that can decompose insecticides would allow the transgenic microorganisms to be used in areas in more extreme temperatures. Several research groups are developing genetically modified bacteria that have enhanced capacity for cleaning areas polluted with heavy metals, radioactive elements, chemical fertilizers, insecticides, herbicides, and other toxic elements (Figure 11-3).

Source: Courtesy of Dr. Julieta Ueta, Pharmaceutical Sciences Department of the College of Pharmaceutical Sciences of Ribeirão Preto, Brazil.

Figure 11-3. Bacteria from soil contaminated with herbicides.

Uranium (U) is the most common contaminant at facilities of the U.S. Department of Energy. The uranyl ion [UO₂]²⁺ is a common, soluble form of this element in the environment. Microbes can immobilize the uranyl ion in several ways, three of which are shown in Figure 11-4. The mineral uraninite (UO₂) is highly insoluble. Microbes can reduce uranyl ion into hydrated uraninite. A cytochrome-c3 hydrogenase from the Desulfovibrio vulgaris bacteria and other organisms can carry out the reduction. Reaction A in the middle branch of Figure 11-4 can be carried out by Deinococcus radiodurans.

Figure 11-4. Uranium biodegradation.

The uranyl ion can also be precipitated as cell-bound hydrogen uranyl phosphate without a change in the oxidation state of the uranium, as shown in the right pathway branch in Figure 11-4. This reaction is facilitated by acid phosphatase N from Citrobacter sp.

Aside from the world of microbes, some plants have also been found to possess the ability to absorb and compartmentalize different elements from the soil, making them useful in bioremediation. At the beginning of the 20th century, it was speculated that some plant species could be used as indicators for the presence of gold in the soil. Populations of certain species were especially abundant in areas rich in the metal.

The mobilization and concentration of metals in plants involves a group of proteins called metalthionins. Some of those proteins are very selective, accumulating specific types of metals. The capability of some plants to absorb metals can be used to extract heavy metals from polluted soil and water.

For a plant to be used in bioremediation, it should do the following:

Plants should absorb and accumulate the metal in an efficient way and must be adapted to a wide range of environmental conditions so that they can be introduced in the most diverse environments where pollution might occur.

After cleaning the polluted area, plants will have absorbed the contaminating element and completed their life cycle. They should then be harvested and removed from the area, and the pollutant present in their tissue should be treated in a way to avoid pollution elsewhere. Biotechnology offers opportunities for improving the capability of mobilization and absorption of metals by plant species by the overexpression of the genes that code for metalthionins. Transferring this trait from bacteria or a plant to a crop species with large adaptability could be one of the major contributions of genetic engineering in bioremediation.